In multiple sclerosis patients, demyelination is prominent in both the white and gray matter. Chronic clinical deficits are known to result from acute or chronic injury to the myelin sheath and inadequate remyelination. The underlying molecular mechanisms of remyelination and its failure remain currently unclear. Recent studies have recognized G protein-coupled receptor 17 (GPR17) as an important regulator of oligodendrocyte development and remyelination. So far, the relevance of GPR17 for myelin repair was mainly tested in remyelinating white matter lesions. The relevance of GPR17 for gray matter remyelination as well as remyelination of chronic white matter lesions was not addressed so far. Here, we provide a detailed characterization of GPR17 expression during experimental de- and remyelination. Experimental lesions with robust and limited endogenous remyelination capacity were established by either acute or chronic cuprizone-induced demyelination. Furthermore, remyelinating lesions were induced by the focal injection of lysophosphatidylcholine (LPC) into the corpus callosum. GPR17 expression was analyzed by complementary techniques including immunohistochemistry, in situ hybridization, and real-time PCR. In control animals, GPR17+ cells were evenly distributed in the corpus callosum and cortex and displayed a highly ramified morphology. Virtually all GPR17+ cells also expressed the oligodendrocyte-specific transcription factor OLIG2. After acute cuprizone-induced demyelination, robust endogenous remyelination was evident in the white matter corpus callosum but not in the gray matter cortex. Endogenous callosal remyelination was paralleled by a robust induction of GPR17 expression which was absent in the gray matter cortex. Higher numbers of GPR17+ cells were as well observed after LPC-induced focal white matter demyelination. In contrast, densities of GPR17+ cells were comparable to control animals after chronic cuprizone-induced demyelination indicating quiescence of this cell population. Our findings demonstrate that GPR17 expression induction correlates with acute demyelination and sufficient endogenous remyelination. This strengthens the view that manipulation of this receptor might be a therapeutic opportunity to support endogenous remyelination.
Literature context: llipore MABN50 (clone 211F1.1), RRID:AB_10807410 Mouse monoclonal anti-GFAP (Cy3
We tested the feasibility and safety of human-spinal-cord-derived neural stem cell (NSI-566) transplantation for the treatment of chronic spinal cord injury (SCI). In this clinical trial, four subjects with T2-T12 SCI received treatment consisting of removal of spinal instrumentation, laminectomy, and durotomy, followed by six midline bilateral stereotactic injections of NSI-566 cells. All subjects tolerated the procedure well and there have been no serious adverse events to date (18-27 months post-grafting). In two subjects, one to two levels of neurological improvement were detected using ISNCSCI motor and sensory scores. Our results support the safety of NSI-566 transplantation into the SCI site and early signs of potential efficacy in three of the subjects warrant further exploration of NSI-566 cells in dose escalation studies. Despite these encouraging secondary data, we emphasize that this safety trial lacks statistical power or a control group needed to evaluate functional changes resulting from cell grafting.
Literature context: # MABN50, RRID:AB_10807410, Billerica
White matter injury (WMI) of prematurity is associated with a spectrum of neurological disorders ranging from mild cognitive and behavioral deficits to cerebral palsy. Translational studies have implicated impaired oligodendrocyte development after hypoxia as the primary cause of WMI, but the underlying mechanisms remain poorly understood. The goal of this study was to identify alterations in the expression of oligodendrocyte precursor cell transcription factors in a mouse model of transient mild global hypoxia. Postnatal day (P) 7 mouse pups were exposed to hypoxia (7.5% O2) for 60minutes. We compared oligodendrocyte differentiation and subsequent myelin formation between hypoxia and sham animals at P9, P14 and P28 by examining the expression of key transcription factor regulators of oligodendrocyte differentiation (Ascl1, Olig1, Olig2, and Nkx2.2), as well as APC, a mature oligodendrocyte marker, in the major white matter regions including the corpus callosum, external capsule and anterior commissure. We also examined the effect on myelin formation by examining two myelin specific protein constituents, myelin associated glycoprotein (MAG) and myelin basic protein (MBP), in white matter tracts and whole brain lysate respectively. We found that transient hypoxia at P7 altered the expression of Ascl1, Olig1 and Nkx2.2, resulting in delayed myelination in the external capsule. In addition, our study showed that oligodendrocyte progenitor cells specified several days prior to a hypoxic event are more susceptible to maturation arrest than those specified shortly prior to hypoxia. Our results suggest that alterations of Ascl1, Olig1 and Nkx2.2 underlie impaired oligodendrocyte differentiation and deficient myelination in WMI. These transcription factors are potential therapeutic targets for the treatment of WMI in preterm infants.
Literature context: chool, Cambridge, Massachusetts RRID:AB_10807410 Rabbit polyclonal anti-Olig2 Mi
Gliomas comprise heterogeneous malignant glial and stromal cells. While blood vessel co-option is a potential mechanism to escape anti-angiogenic therapy, the relevance of glial phenotype in this process is unclear. We show that Olig2+ oligodendrocyte precursor-like glioma cells invade by single-cell vessel co-option and preserve the blood-brain barrier (BBB). Conversely, Olig2-negative glioma cells form dense perivascular collections and promote angiogenesis and BBB breakdown, leading to innate immune cell activation. Experimentally, Olig2 promotes Wnt7b expression, a finding that correlates in human glioma profiling. Targeted Wnt7a/7b deletion or pharmacologic Wnt inhibition blocks Olig2+ glioma single-cell vessel co-option and enhances responses to temozolomide. Finally, Olig2 and Wnt7 become upregulated after anti-VEGF treatment in preclinical models and patients. Thus, glial-encoded pathways regulate distinct glioma-vascular microenvironmental interactions.
Literature context: ody (Millipore catalog #MABN50 RRID:AB_10807410, 1:1000); rabbit anti-Olig2 ant
NG2 cells are a resident glial progenitor cell population that is uniformly distributed throughout the developing and mature mammalian CNS. Those in the postnatal CNS generate exclusively myelinating and non-myelinating oligodendrocytes and are thus equated with oligodendrocyte precursor cells. Prenatally, NG2 cells in the ventral gray matter of the forebrain generate protoplasmic astrocytes as well as oligodendrocytes. The fate conversion from NG2 cells into protoplasmic astrocytes is dependent on downregulation of the key oligodendrocyte transcription factor Olig2. We showed previously that constitutive deletion of Olig2 in NG2 cells converts NG2 cells in the neocortex into protoplasmic astrocytes at the expense of oligodendrocytes. In this study, we show that postnatal deletion of Olig2 caused NG2 cells in the neocortex but not in other gray matter regions to become protoplasmic astrocytes. However, NG2 cells in the neocortex became more resistant to astrocyte fate switch over the first 3 postnatal weeks. Fewer NG2 cells differentiated into astrocytes and did so with longer latency after Olig2 deletion at postnatal day 18 (P18) compared with deletion at P2. The high-mobility group transcription factor Sox10 was not downregulated for at least 1 month after Olig2 deletion at P18 despite an early transient upregulation of the astrocyte transcription factor NFIA. Furthermore, inhibiting cell proliferation in slice culture reduced astrocyte differentiation from Olig2-deleted perinatal NG2 cells, suggesting that cell division might facilitate nuclear reorganization needed for astrocyte transformation.SIGNIFICANCE STATEMENT NG2 cells are glial progenitor cells that retain a certain degree of lineage plasticity. In the normal postnatal neocortex, they generate mostly oligodendrocyte lineage cells. When the oligodendrocyte transcription factor Olig2 is deleted in NG2 cells in the neocortex, they switch their fate to protoplasmic astrocytes. However, the efficiency of the fate switch decreases with age over the first 3 postnatal weeks and is reduced when cell proliferation is inhibited. As the neocortex matures, sustained expression of the oligodendrocyte lineage-specific key transcription factor Sox10 becomes less dependent on Olig2. Together, our findings suggest a gradual stabilization of the oligodendrocyte lineage genes and loss of lineage plasticity during the first 3 weeks after birth, possibly due to nuclear reorganization.
Literature context: Millipore, order numb. MABN50, RRID:AB_10807410) combined with anti-ATF3 (1 : 2
The extent of remyelination in multiple sclerosis lesions is often incomplete. Injury to oligodendrocyte progenitor cells can be a contributing factor for such incomplete remyelination. The precise mechanisms underlying insufficient repair remain to be defined, but oxidative stress appears to be involved. Here, we used immortalized oligodendrocyte cell lines as model systems to investigate a causal relation of oxidative stress and endoplasmic reticulum stress signaling cascades. OLN93 and OliNeu cells were subjected to chemical hypoxia by blocking the respiratory chain at various levels. Mitochondrial membrane potential and oxidative stress levels were quantified by flow cytometry. Endoplasmic reticulum stress was monitored by the expression induction of activating transcription factor 3 and 4 (Atf3, Atf4), DNA damage-inducible transcript 3 protein (Ddit3), and glucose-regulated protein 94. Lentiviral silencing of nuclear factor (erythroid-derived 2)-like 2 or kelch-like ECH-associated protein 1 was applied to study the relevance of NRF2 for endoplasmic reticulum stress responses. We demonstrate that inhibition of the respiratory chain induces oxidative stress in cultured oligodendrocytes which is paralleled by the expression induction of distinct mediators of the endoplasmic reticulum stress response, namely Atf3, Atf4, and Ddit3. Atf3 and Ddit3 expression induction is potentiated in kelch-like ECH-associated protein 1-deficient cells and absent in cells lacking the oxidative stress-related transcription factor NRF2. This study provides strong evidence that oxidative stress in oligodendrocytes activates endoplasmic reticulum stress response in a NRF2-dependent manner and, in consequence, might regulate oligodendrocyte degeneration in multiple sclerosis and other neurological disorders.
Literature context: RRID:AB_10807410; Rabbit anti-MBP, Cat# AB_980;
The oligodendrocyte density is greater and myelin sheaths are thicker in the adult male mouse brain when compared with females. Here, we show that these sex differences emerge during the first 10 postnatal days, precisely at a stage when a late wave of oligodendrocyte progenitor cells arises and starts differentiating. Androgen levels, analyzed by gas chromatography/tandem-mass spectrometry, were higher in males than in females during this period. Treating male pups with flutamide, an androgen receptor (AR) antagonist, or female pups with 5α-dihydrotestosterone (5α-DHT), revealed the importance of postnatal androgens in masculinizing myelin and their persistent effect into adulthood. A key role of the brain AR in establishing the sexual phenotype of myelin was demonstrated by its conditional deletion. Our results uncover a new persistent effect of postnatal AR signaling, with implications for neurodevelopmental disorders and sex differences in multiple sclerosis.
Literature context: 50, Millipore; RRID:AB_10807410).
Currently no treatments exist for preterm infants with diffuse white matter injury (DWMI) caused by hypoxia. Due to improved care of preterm neonates and increased recognition by advanced imaging techniques, the prevalence of DWMI is increasing. A better understanding of the pathophysiology of DWMI is therefore of critical importance. The integrated stress response (ISR), a conserved eukaryotic response to myriad stressors including hypoxia, may play a role in hypoxia-induced DWMI and may represent a novel target for much needed therapies. In this study we utilize in vitro and in vivo hypoxic models of DWMI to investigate whether the ISR is involved in DWMI. We demonstrate that hypoxia activates the ISR in primary mouse oligodendrocyte precursor cells (OPCs) in vitro and that genetically inhibiting the ISR in differentiating OPCs increases their susceptibility to in vitro hypoxia. We also show that a well-established in vivo mild chronic hypoxia (MCH) mouse model and a new severe acute hypoxia (SAH) mouse model of DWMI activates the initial step of the ISR. Nonetheless, genetic inhibition of the ISR has no detectable effect on either MCH or SAH-induced DWMI. In addition, we demonstrate that genetic enhancement of the ISR does not ameliorate MCH or SAH-induced DWMI. These studies suggest that while the ISR protects OPCs from hypoxia in vitro, it does not appear to play a major role in either MCH or SAH-induced DWMI and is therefore not a likely target for therapies aimed at improving neurological outcome in preterm neonates with hypoxia-induced DWMI.SIGNIFICANCE STATEMENTDiffuse white matter injury (DWMI) caused by hypoxia is a leading cause of neurological deficits following premature birth. An increased understanding of the pathogenesis of this disease is critical. The integrated stress response (ISR) is activated by hypoxia and protects oligodendrocyte lineage cells in other disease models. This has led to an interest in the potential role of the ISR in DWMI. Here we examine the ISR in hypoxia-induced DWMI and show that while the ISR protects oligodendrocyte lineage cells from hypoxia in vitro, genetic inhibition or enhancement of the ISR has no effect on hypoxia-induced DWMI in vivo suggesting that the ISR does not play a major role in, and is not a likely therapeutic target for, DWMI.
Literature context: g #MABN50 RRID:AB-10807410, 1:200), a
Diencephalic defects underlie an array of neurological diseases. Previous studies have suggested that retinoic acid (RA) signaling is involved in diencephalic development at late stages of embryonic development, but its roles and mechanisms of action during early neural development are still unclear. Here we demonstrate that mice lacking enzymatic activity of the acetyltransferase GCN5 ((Gcn5hat/hat )), which were previously characterized with respect to their exencephalic phenotype, exhibit significant diencephalic expansion, decreased diencephalic RA signaling, and increased diencephalic WNT and SHH signaling. Using a variety of molecular biology techniques in both cultured neuroepithelial cells treated with a GCN5 inhibitor and forebrain tissue from (Gcn5hat/hat ) embryos, we demonstrate that GCN5, RARα/γ, and the poorly characterized protein TACC1 form a complex in the nucleus that binds specific retinoic acid response elements in the absence of RA. Furthermore, RA triggers GCN5-mediated acetylation of TACC1, which results in dissociation of TACC1 from retinoic acid response elements and leads to transcriptional activation of RA target genes. Intriguingly, RA signaling defects caused by in vitro inhibition of GCN5 can be rescued through RA-dependent mechanisms that require RARβ. Last, we demonstrate that the diencephalic expansion and transcriptional defects seen in (Gcn5hat/hat ) mutants can be rescued with gestational RA supplementation, supporting a direct link between GCN5, TACC1, and RA signaling in the developing diencephalon. Together, our studies identify a novel, nonhistone substrate for GCN5 whose modification regulates a previously undescribed, tissue-specific mechanism of RA signaling that is required to restrict diencephalic size during early forebrain development.SIGNIFICANCE STATEMENT Changes in diencephalic size and shape, as well as SNPs associated with retinoic acid (RA) signaling-associated genes, have been linked to neuropsychiatric disorders. However, the mechanisms that regulate diencephalic morphogenesis and the involvement of RA signaling in this process are poorly understood. Here we demonstrate a novel role of the acetyltransferase GCN5 in a previously undescribed mechanism of RA signaling in the developing forebrain that is required to maintain the appropriate size of the diencephalon. Together, our experiments identify a novel nonhistone substrate of GCN5, highlight an essential role for both GCN5 and RA signaling in early diencephalic development, and elucidate a novel molecular regulatory mechanism for RA signaling that is specific to the developing forebrain.
Literature context: # MABN50, RRID:AB_10807410 Mouse mono
Huntingtin-associated protein 1 (HAP1) is a neuronal interactor with causatively polyglutamine (polyQ)-expanded huntingtin in Huntington's disease and also associated with pathologically polyQ-expanded androgen receptor (AR) in spinobulbar muscular atrophy (SBMA), being considered as a protective factor against neurodegenerative apoptosis. In normal brains, it is abundantly expressed particularly in the limbic-hypothalamic regions that tend to be spared from neurodegeneration, whereas the areas with little HAP1 expression, including the striatum, thalamus, cerebral neocortex and cerebellum, are targets in several neurodegenerative diseases. While the spinal cord is another major neurodegenerative target, HAP1-immunoreactive (ir) structures have yet to be determined there. In the current study, HAP1 expression was immunohistochemically evaluated in light and electron microscopy through the cervical, thoracic, lumbar, and sacral spinal cords of the adult male rat. Our results showed that HAP1 is specifically expressed in neurons through the spinal segments and that more than 90% of neurons expressed HAP1 in lamina I-II, lamina X, and autonomic preganglionic regions. Double-immunostaining for HAP1 and AR demonstrated that more than 80% of neurons expressed both in laminae I-II and X. In contrast, HAP1 was specifically lacking in the lamina IX motoneurons with or without AR expression. The present study first demonstrated that HAP1 is abundantly expressed in spinal neurons of the somatosensory, viscerosensory, and autonomic regions but absent in somatomotor neurons, suggesting that the spinal motoneurons are, due to lack of putative HAP1 protectivity, more vulnerable to stresses in neurodegenerative diseases than other HAP1-expressing neurons probably involved in spinal sensory and autonomic functions.
To determine whether L-type voltage-operated Ca2+ channels (L-VOCCs) are required for oligodendrocyte progenitor cell (OPC) development, we generated an inducible conditional knock-out mouse in which the L-VOCC isoform Cav1.2 was postnatally deleted in NG2-positive OPCs. A significant hypomyelination was found in the brains of the Cav1.2 conditional knock-out (Cav1.2KO) mice specifically when the Cav1.2 deletion was induced in OPCs during the first 2 postnatal weeks. A decrease in myelin proteins expression was visible in several brain structures, including the corpus callosum, cortex, and striatum, and the corpus callosum of Cav1.2KO animals showed an important decrease in the percentage of myelinated axons and a substantial increase in the mean g-ratio of myelinated axons. The reduced myelination was accompanied by an important decline in the number of myelinating oligodendrocytes and in the rate of OPC proliferation. Furthermore, using a triple transgenic mouse in which all of the Cav1.2KO OPCs were tracked by a Cre reporter, we found that Cav1.2KO OPCs produce less mature oligodendrocytes than control cells. Finally, live-cell imaging in early postnatal brain slices revealed that the migration and proliferation of subventricular zone OPCs is decreased in the Cav1.2KO mice. These results indicate that the L-VOCC isoform Cav1.2 modulates oligodendrocyte development and suggest that Ca2+ influx mediated by L-VOCCs in OPCs is necessary for normal myelination. SIGNIFICANCE STATEMENT: Overall, it is clear that cells in the oligodendrocyte lineage exhibit remarkable plasticity with regard to the expression of Ca2+ channels and that perturbation of Ca2+ homeostasis likely plays an important role in the pathogenesis underlying demyelinating diseases. To determine whether voltage-gated Ca2+ entry is involved in oligodendrocyte maturation and myelination, we used a conditional knock-out mouse for voltage-operated Ca2+ channels in oligodendrocyte progenitor cells. Our results indicate that voltage-operated Ca2+ channels can modulate oligodendrocyte development in the postnatal brain and suggest that voltage-gated Ca2+ influx in oligodendroglial cells is critical for normal myelination. These findings could lead to novel approaches to intervene in neurodegenerative diseases in which myelin is lost or damaged.
Literature context: o human Olig2Millipore, MABN50, AB_10807410, mouse, monoclonal1:250Anti-Sox
Two waves of oligodendrogenesis in the ventricular zone of the spinal cord (SC-VZ) during rat development, which take place between embryonic days 14 and 18 (E14-E18) and E20-E21, have been described. In the VZ of the brain, unlike the SC-VZ, a third wave of oligodendrogenesis occurs during the first weeks of postnatal development. Using immunofluorescence staining of intact rat SC tissue, we noticed the presence of small numbers of Olig2(+) /Sox-10(+) cells inside the lining of the central canal (CC) during postnatal development and adulthood. Olig2(+) /Sox-10(+) cells appeared inside the lining of the CC shortly after birth, and their number reached a maximum of approximately 0.65 ± 0.14 cell/40-μm section during the second postnatal week. After the latter development, the number of Olig2(+) /Sox-10(+) cells decreased to 0.21 ± 0.07 (P36) and 0.18 ± 0.1 cell/section (P120). At P21, Olig2(+) /Sox-10(+) cells inside the CC lining started to express other oligodendroglial markers such as CNPase, RIP, and APC. Olig2(+) /Sox-10(+) cells usually did not proliferate inside the CC lining and were only rarely found to be immunoreactive against oligodendrocyte progenitor markers such as NG2 or PDGFRα. Using 5-bromo-2-deoxyuridine administration at P2, P11, P22, or P120-P125, we revealed that these cells arose in the CC lining during postnatal development and adulthood. Our findings confirmed that the CC lining is the source of a small number of cells with an oligodendroglial phenotype during postnatal development and adulthood in the SC of intact rats.
Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder with variable expressivity. Heterozygous mutations in either of two genes, TSC1 (hamartin) or TSC2 (tuberin), are responsible for most cases. Hamartin and tuberin form a heterodimer that functions as a major cellular inhibitor of the mammalian target of rapamycin complex 1 (mTORC1) kinase. Genotype-phenotype studies suggest that TSC2 mutations are associated with a more severe neurologic phenotype, although the biologic basis for the difference between TSC1- and TSC2-based disease is unclear. Here we performed a study to compare and contrast the brain phenotypes of Tsc1 and Tsc2 single and double mutants. Using Tsc1 and Tsc2 floxed alleles and a radial glial transgenic Cre driver (FVB-Tg(GFAP-cre)25Mes/J), we deleted Tsc1 and/or Tsc2 in radial glial progenitor cells. Single and double mutants had remarkably similar phenotypes: early postnatal mortality, brain overgrowth, laminar disruption, astrogliosis, a paucity of oligodendroglia, and myelination defects. Double Tsc1/Tsc2 mutants died earlier than single mutants, and single mutants showed differences in the location of heterotopias and the organization of the hippocampal stratum pyramidale. The differences were not due to differential mTORC1 activation or feedback inhibition on Akt. These data provide further genetic evidence for individual hamartin and tuberin functions that may explain some of the genotype-phenotype differences seen in the human disease.